Antimicrobial peptides, particularly cationic peptides have received increasing attention as a new pharmaceutical substance, because of their broad spectrum of antimicrobial activities and the rapid development of multi-drug-resistant pathogenic microorganisms. Endogenous peptide antibiotics are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. These endogenous antimicrobial peptides are usually cationic amphipathic molecules that contain 10 to 45 amino acid residues and an excess of lysine and arginine residues. (for a review, see Broekaert et al., Plant Physiol. 108:1353, 1995; Ganz and Lehrer, Pharmacol. Ther. 66:191, 1995; Martin et al., J. Leukoc. Biol. 58:128, 1995; Hancock and Lehrer, TIBTECH 16:82, 1998). Examples of cationic peptides include rabbit defensin, crab tachyplesin, bovine bactenecin, silk-moth cecropin A, frog magainins, and bovine indolicidin. The main site of action of the peptides is the cytoplasmic membrane of bacteria and other microbes. Due to their amphipathic nature, the peptides disrupt the membrane, causing a loss of potassium ions, membrane depolarization, and a decrease in cytoplasmic ATP.
Since their de novo synthesis or release from storage sites can be induced rapidly, cationic peptides are particularly important in the initial phases of resistance to microbial invasion. Cationic peptides are also effective when administered as therapeutic agents. In the treatment of topical infection, for example, an α-helical magainin variant peptide has been shown to be effective against polymicrobic foot ulcer infections in diabetics, and a protegrin-derived peptide was found useful for treatment of oral polymicrobic ulcers in cancer patients (Hancock and Lehrer, TIBTECH 16:82, 1998). Efficacy against systemic infection has been shown with an α-helical peptide used to treat Pseudomonas aeruginosa peritoneal infection, a β-sheet protegrin against methicillin-resistant Staphylococcus aureus and against vancomycin-resistant Enterococcus faecalis, and extended-helix indolicidin against Aspergillus fungal infections (Gough et al., Infect. Immun. 64:4922, 1996; Steinberg et al., Antimicrob. Agents Chemother. 41:1738, 1997; and Ahmad et al, Biochim. Biophys. Acta 1237:109, 1995). Therefore, naturally-occurring cationic peptides, and their synthetic variants, are valuable antimicrobial therapeutics.
A practical drawback in cationic peptide therapy is the lack of a cost effective, mass-production method of the agents. Typically, the isolation of cationic peptides from natural sources is not cost-effective, and does not apply to the production of engineered cationic peptide variants which may have increased efficacy. While chemical peptide synthesis can be used to manufacture either natural or engineered cationic peptides, this approach is very costly.
Therefore, alternate, more economical and efficient methods of synthesis are needed, such as in vivo synthesis in host cells using recombinant DNA methods. Researchers have attempted various methods for recombinant production of cationic peptides. For example, cationic peptides have been produced in bacteria, such as E. coli or Staphylococcus aureus, yeast, insect cells, and transgenic mammals (Piers et al., Gene 134:7, 1993, Reichhart et al., Invertebrate Reprod. Develop. 21:15, 1992, Hellers et at, Eur. J. Biochem. 199:435, 1991, and Sharma et al, Proc. Nat'l Acad Sci. USA 91:9337, 1994).
Much attention has focused on production in E. coli, since those versed in the art are familiar with the fact that high productivity can be obtained in E. coli using the recombinant DNA technology. However, for small peptides it is often necessary to produce them as part of a larger fusion protein. In this technique the gene for the peptide is joined to that of a larger carrier protein and the fusion expressed as a single larger protein. Following synthesis the peptide must be cleaved from the fusion partner. There is an extensive body of literature on protein fusion, especially in the gene expression host E. coli. For example, a number of recombinant proteins have been produced as fusion proteins in E. coli, such as, insulin A and B chain, calcitonin, Beta-globin, myoglobin, and a human growth hormone (Uhlen and Moks, “Gene Fusions for Purposes of Expression, An Introduction” in Methods in Enzymology 185:129-143 Academic Press, Inc. 1990). Nevertheless, recombinant gene expression from a host cell presents a number of technical problems, particularly if it is desired to produce large quantities of a particular protein. For example, if the protein is a cationic peptide, such peptides are very susceptible to proteolytic degradation, possibly due to their small size or lack of highly ordered tertiary structure. One approach to solving this problem is to produce recombinant cationic proteins in protease-deficient E. coli host cell strains (see, for example, Williams et al., U.S. Pat. No. 5,589,364, and WO 96/04373). Yet there is no general way to predict which protease-deficient strains will be effective for a particular recombinant protein.
In principle the recombinant DNA technique is straight forward. However, any sequence that interferes with bacterial growth through replication or production of products toxic to the bacteria, such as lytic cationic peptides, are problematic for cloning. Foreign peptide gene products that are unstable or toxic, like cationic peptides, can also be stabilized by expressing the peptides as part of a fusion protein comprising a host cell protein. For example, Callaway et al. et al., Antimicrob. Agents Chemother. 37:1614, 1993, expressed cecropin A in E. coli as a fusion peptide with a truncated portion of the L-ribulokinase gene product, Piers et al. et al., Gene 134:7, 1993, expressed fusion proteins in E. coli that comprised glutathione-S-transferase and either defensin (HNP-1) or a synthetic cecropin-melittin hybrid, while Hara et al., Biochem. Biophys. Res. Commun. 220:664, 1996, expressed silkworm moricin in E. coli as a fusion protein with a β-galactosidase or a maltose-binding protein moiety.
One of the better options to avoid the toxic effects of a bacteriolytic peptide on the host bacterial cells in highly efficient production, and to avoid proteolytic degradation of the peptides, is to utilize the intrinsic bacterial host mechanism of driving heterologous proteins into inclusion bodies as a denatured insoluble form.
The approach outlined above suffers from the inherent limitation on overall productivity imposed by the use of a small single peptide (circa 10%) in the large fusion protein.
Accordingly, a need exists for a means to efficiently produce cationic peptides from recombinant host cells.